ABSTRACT. This grant examines the participation of the dihydropyridine receptor (DHPR) beta1a and beta2a subunits in skeletal and cardiac muscle excitation-contraction (EC) coupling. In response to depolarization, the DHPR products a signal that briefly opens ryanodine receptor (RyR) channels leading toe the release of stored Ca2+. Considerable progress in the understanding DHPR-RyR interactions has been made by the availability of mouse models for the expression of DHPRs and RyRs. These are the dysgenic mouse line lacking alpha2S and knockout mice lacking the skeletal muscle RyR1 isoform or lacking the beta1a subunit of the skeletal muscle DHPR. The proposed experiments will use these mutants to identify domains of the beta1a and beta2a subunits that participate in EC coupling. AC-terminus region of beta1a, unrelated to the BID domain required for binding to alpha1S, will be characterized in detail. This C-terminus region of beta1a, unrelated to the BID domain required for binding to alpha1S, will be characterized in detail. This C- terminus could bring about a stronger colocalization of DHPRs and RyRs or could be essential for the generation of the signal that opens the RyR. Both possibilities will be tested. To address these questions, we make extensive use of double-null myotubes, (alpha1S/beta)-null and (beta1/RyR1)-null, generated by mouse breeding. Double-null skeletal muscle cells should permit studies of alpha2S/beta and beta/RyR interactions in expression systems in which the two missing subunits can be expressed and modified. The specific aims of the application are: Aim 1. Establish the role of beta1a and beta2a in the expression of DHPRs specifically required for EC coupling; Aim 2. Identify molecular domains of beta1a required for EC coupling; Aim 3. Test functional beta-RyR interactions controlling Ca2+ sparks; and Aim 4. Determine whether beta triggers a component of the Ca2+ transient. The latter is investigated by expression of alpha1S constructs lacking the II/III loop and by expression of alpha1C constructs in ES cell-derived cardiomyocytes. The main methods include a) expression of cDNA constructs of alpha1, beta and RyR subunits in single subunit-deficient or in double-null myotubes in culture; b) transgenic over-expression of beta1 constructs; c) expression of alpha1C constructs in alpha1C-null cardiomyocytes; d) macroscopic measurements of Ca2+ currents and charge movements in voltage- clamped cells; and e) confocal imaging of Ca2+ transients and Ca2+ sparks. DHPR-RyR interactions are crucial for understanding the molecular basis of EC coupling in normal and diseased states of skeletal and cardiac muscle.